Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

Abstract

he SARS-CoV-2 virus is thought, based on sequence identity, to have crossed from bats to humans in 2019 1 . Similar to SARS-CoV-1 (2002-2003 and MERS-CoV (2012), SARS-CoV-2 presents as a respiratory disease but can progress into internal organs and cause organ failure 2,3 . A recent report from France estimates a fatality rate of 0.7% and a hospitalization rate of 3.6% 4 . Both these rates are much higher in elderly populations 4,5 . Around 33% of those admitted to UK hospitals with COVID-19 have died 6 . Because SARS-CoV-2 also spreads rapidly in the naive human population 7 , the current COVID-19 pandemic has presented an unprecedented challenge to modern human society. Although there is currently no 'cure' or vaccine for the disease, passive immune therapy by transfusing critically ill COVID-19 patients with serum from COVID-19 convalescent individuals has been shown to improve clinical outcomes 8,9 . This would suggest that neutralization of the virus, even at a relatively late stage in the disease, may be a useful COVID-19 therapy.The single-positive-strand RNA genome of SARS-CoV-2, like SARS-CoV, encodes four major structural proteins: spike, envelope, membrane and nucleocapsid. The spike protein comprises an N-terminal (S1) subunit, which contains the roughly 200-residue receptor binding domain (RBD) 10,11 , and a C-terminal subunit (S2), which contains the fusion protein 12 (Fig. 1a). The RBD of SARS-CoV-2 binds more tightly to the extracellular domain of angiotensin-converting enzyme 2 (ACE2) (Fig. 1a) than the homologous SARS-CoV-1 RBD 13 . The higher affinity results from sequence changes in RBD (Fig. 1b) and this has been proposed to underlie the higher transmissibility of SARS-CoV-2 14 . Antibodies raised to the spike protein of SARS-CoV-1 can neutralize the virus both in vitro and in vivo, by binding to the RBD and blocking binding to ACE2 15 . Unfortunately, most of these antibodies do not cross-react with the SARS-CoV-2 RBD 13 . The CR3022 antibody derived from a convalescent SARS-CoV-1 patient is cross-reactive to both SARS-CoV-1 and SARS-CoV-2 RBD (reported apparent K D of 6 nM, ref. 16 ). Two studies have reported crystal structures of CR3022 bound to SARS-CoV-2 RBD and show that the target epitope is distant from the ACE2 binding region 17,18 , which is consistent with the observation that CR3022 does not block RBD binding to ACE2. Another study on CR3022 has reported highly effective SARS-CoV-2 neutralizing activity that appears to arise from destabilization of the spike trimer, a novel mechanism for neutralizing SARS-CoV-2 18 . Destabilization of viral proteins by antibodies has been observed for influenza 19 and human immunodeficiency virus 20 .Mammalian, including human, antibodies generally have two chains (heavy and light), but camelids, in addition to two-chain antibodies, also possess a single-heavy-chain antibody variant 21 .

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

“…Thus, when mNb6-tri engages with Spike, it prevents ACE2 binding by both directly occluding the binding site and by locking the RBDs into an inactive conformation. Although a multitude of other monoclonal and single-domain antibodies against SARS-CoV-2 Spike have been discovered to date, there are few if any molecules as potent and stable as mNb6-tri (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43).…”

Breadth and function of antibody response to acute SARS-CoV-2 infection in humans

“…We compared two assays designed to detect antibody-blocking of the ACE2-RBD interaction (Supplementary Figure 5, Table 1), including an assay that we reported previously (13,16). In the first assay unlabelled MAbs in at least 10-fold excess were mixed with biotin labelled ACE2-Fc and binding to the RBD, displayed on RBD-VLP bound to an ELISA plate, was detected with Streptavidin-HRP (Supplementary Figure 5a).…”

“…In the first ACE2 blocking assay, RBD-VLP (Spycatcher-mi3 VLP-particles conjugated with Spytagged-RBD recombinant protein) (17) was coated on ELISA plates as described for the competitive binding assay. Recombinant ACE2-Fc protein expressed in Expi293F (Life Technologies) The second ACE2 blocking assay was performed as described previously (13,16). A n # -R B D , 1 , 2 % A n # -S 1 ( n o n R B D ) , 1 , 2 % A n # -S 2 , 1 , 2 % A n # -N , 7 , 1 4 % U n d e t e r m i n e d , 4 0 , 8 0 % A n # -S p i k e , 3 , 6…”

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

“…Most neutralizing antibodies to SARS-CoV-1 and CoV-2 block viral entry by binding to the ACE2 binding site on the RBD. Ongoing efforts by our lab and others use in vitro methods, such as phage display or yeast display, from naïve libraries to generate recombinant antibodies or other formatted domains to block viral entry (9,10).…”